DEAD‐Box Helicase 17 exacerbates non‐alcoholic steatohepatitis via transcriptional repression of cyp2c29, inducing hepatic lipid metabolism disorder and eliciting the activation of M1 macrophages

Abstract Objective Our study was to elucidate the role of RNA helicase DEAD‐Box Helicase 17 (DDX17) in NAFLD and to explore its underlying mechanisms. Methods We created hepatocyte‐specific Ddx17‐deficient mice aim to investigate the impact of Ddx17 on NAFLD induced by a high‐fat diet (HFD) as well as methionine and choline‐deficient l‐amino acid diet (MCD) in adult male mice. RNA‐seq and lipidomic analyses were conducted to depict the metabolic landscape, and CUT&Tag combined with chromatin immunoprecipitation (ChIP) and luciferase reporter assays were conducted. Results In this work, we observed a notable increase in DDX17 expression in the livers of patients with NASH and in murine models of NASH induced by HFD or MCD. After introducing lentiviruses into hepatocyte L02 for DDX17 knockdown or overexpression, we found that lipid accumulation induced by palmitic acid/oleic acid (PAOA) in L02 cells was noticeably weakened by DDX17 knockdown but augmented by DDX17 overexpression. Furthermore, hepatocyte‐specific DDX17 knockout significantly alleviated hepatic steatosis, inflammatory response and fibrosis in mice after the administration of MCD and HFD. Mechanistically, our analysis of RNA‐seq and CUT&Tag results combined with ChIP and luciferase reporter assays indicated that DDX17 transcriptionally represses Cyp2c29 gene expression by cooperating with CCCTC binding factor (CTCF) and DEAD‐Box Helicase 5 (DDX5). Using absolute quantitative lipidomics analysis, we identified a hepatocyte‐specific DDX17 deficiency that decreased lipid accumulation and altered lipid composition in the livers of mice after MCD administration. Based on the RNA‐seq analysis, our findings suggest that DDX17 could potentially have an impact on the modulation of lipid metabolism and the activation of M1 macrophages in murine NASH models. Conclusion These results imply that DDX17 is involved in NASH development by promoting lipid accumulation in hepatocytes, inducing the activation of M1 macrophages, subsequent inflammatory responses and fibrosis through the transcriptional repression of Cyp2c29 in mice. Therefore, DDX17 holds promise as a potential drug target for the treatment of NASH.


Graphical Abstract
• DDX17 expression is elevated in the livers of patients with NASH.
• DDX17 accelerates NASH development by promoting lipid accumulation in hepatocytes.
• DDX17 alters lipid composition in murine NASH model.
• Hepatocyte DDX17 induces the activation of M1 macrophages and subsequent inflammatory response and fibrosis through the transcriptional repression of Cyp2c29 in mice.
• Cyp2c29 expression is decreased in the liver of NASH models and meditates the function of DDX17. Clin.Transl.Med.2024;14:e1529.
wileyonlinelibrary.com/journal/ctm2 https://doi.org/10.1002/ctm2.15292] NAFLD covers a broad range of diseases, spanning from steatosis to non-alcoholic steatohepatitis (NASH), liver fibrosis and hepatocellular carcinoma (HCC).Due to various risk factors, including genetic mutations, metabolic disorders and chemical injury, NAFLD is characterised as a heterogeneous disease with different mechanisms and progression.Although many studies have been conducted to assist in early diagnosis and to reveal specific mechanisms and therapeutic methods, effective pharmaceuticals are still lacking. 3Thus, further studies on its pathogenesis, mechanisms, animal models and specific treatments are required to provide molecular targets and treatment strategies for clinical use. 4 DEAD-box Helicase 17 (DDX17), also named p72, RNA helicase and its paralog, DDX5, also named p68, belong to the DEAD-box family and function in most steps of gene expression. 5Except as RNA helicases, they also play essential roles in alternative splicing (AS) and transcriptional regulation.It has also been reported that DDX17 and DDX5 promote the invasiveness of tumour through the regulation of AS in several DNA-and chromatin-binding factors, such as macroH2A1 histones. 6RELA recruits DDX17 by activating the NF-κB signalling pathway. 7Recently, DDX17 has been reported to contribute to inflammation as well as tumour initiation and progression by regulating AS. 8,9 Furthermore, DDX17 and DDX5 dynamically orchestrate splicing and transcription by cooperating with several key factors, such as heterogeneous nuclear ribonucleoprotein (hnRNAP) H/F splicing factors 10 and oestrogen and androgen receptors (ER and AR). 11Studies have shown that DDX17 and DDX5 play critical roles in transcription with a series of classic transcription factors (TF), such as ERα 12 and nuclear factor of activated T Cells 5 (NFAT5). 13hey have also been demonstrated to have significant functions in transcription, acting either as coactivators or corepressors by interacting with essential elements of the transcriptional apparatus like RNA polymerase II, histone deacetylases and CREB-binding protein (CBP)/p300. 5nterestingly, DDX17 also assists RE1-silencing TF (REST) in transcriptional repression activity by promoting its interaction with the promoter of a portion of REST-target genes. 14In addition, several recent studies have shown that DDX17 promotes HCC and metastasis. 8,15,16However, few studies have explored the function of DDX17 in cell metabolism and related diseases, with the exception of one study that demonstrated DDX17-mediated transcriptional regulation of macrophage cholesterol efflux during atherogenesis. 17We previously found that DDX17 exhibited an increased expression in the liver or HCC tissues of diethylnitrosamine (DEN)-treated C57 mice fed-with highfat diet (HFD) for 8 months compared with those treated with DEN and fed with normal diet (ND). 18Thus, we hypothesised that DDX17 is involved in the progression of NASH and NASH-HCC; nonetheless, the specific mechanism of DDX17 in lipid metabolism and NASH remain to be thoroughly clarified.
This study highlights a notable increase in DDX17 expression in the livers of individuals diagnosed with NASH and in murine NASH models.We found that, mechanistically, depleting DDX17 in hepatocytes alleviates hepatic steatosis, inflammation and fibrosis in mouse models of NASH induced by both HFD and methionine and choline-deficient l-amino acid diet (MCD), while the overexpression of DDX17 in hepatocytes markedly promoted lipid accumulation.Our analysis of RNA-seq and CUT&Tag, combined with chromatin immunoprecipitation (ChIP) and luciferase reporter assays indicated that DDX17 transcriptionally repressed Cyp2c29 gene expression by cooperating with CCCTC binding factor (CTCF) and DEAD-Box Helicase 5 (DDX5).Using absolute quantitative lipidomics analysis, we found a hepatocytespecific DDX17 deficiency decreased lipid accumulation and altered lipid composition in the livers of mice after MCD administration.Furthermore, combined with RNAseq analysis, lipidome analysis and rescue experiments, we found that DDX17 potentially contributes to the control of lipid metabolism and activation of M1 macrophages by regulating the expression of Cyp2c29 in murine NASH models (Additional files 1).Collectively, our study reveals a novel mechanism and offers promising targets for the treatment of hepatic steatosis, inflammation and fibrosis.

Clinical specimens
Human liver tissues were acquired from individuals without steatosis, with NAFLD or NASH, who had undergone a liver transplantation.The detailed flowchart is presented in Additional files 2. Normal liver, NAFLD and NASH were diagnosed by two pathologists based on the scoring system established by the NASH Clinical Research Network. 19he normal liver was defined as cases with a NASH activity score (NAS) of 0. Simple steatosis was determined by NAS of 1−2, a ballooning score of 0 and the absence of fibrosis.NASH was defined as the cases with NAS ≥ 5 or the presence of fibrosis, NAS of 3−4.Clinical and histological characteristics of the patients are presented in Table S1.Subjects or the immediate families of liver donors provided written informed consent.The research protocols related to human samples received ethical clearance from the Review Board at Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China, and adhered to the ethical principles set forth in the Declaration of Helsinki.

Cell culture
293T cells, L02 cells and RAW264.7 macrophages were cultured in an environment with 5% CO 2 , DMEM containing 10% foetal bovine serum (FBS) and 1% penicillinstreptomycin for their growth.Murine AML12 hepatocytes were cultured in DMEM/F12 supplemented with 10% FBS, 40 ng/mL dexamethasone and 1% ITS (Table S3).The cell lines utilised in our study were obtained from the China Center for Type Culture Collection, and we verified their authenticity via STR analysis.

Induction of the lipid accumulation in hepatocytes
L02 hepatocytes were cultured in a suitable medium, and then they were exposed to palmitic acid and oleic acid (PAOA) at specified concentrations in a 1:2 ratio for a duration of 12 h.We used bovine serum albumin (BSA) without fatty acids as the control.

Plasmid construction, transfections and stable cell line construction
Related plasmids used in this study were constructed according to Vazyme 's homologous recombination method (Cat: C112).In brief, the linearised vector was obtained by digesting the pCDNA3.1−circular vector with indicated resection enzymes.DDX17, DDX5 and CTCF genes were cloned into the pcDNA3.1−vector after respectively amplifying by PCR.And before transfection, all constructs were verified through DNA sequencing.Prior to transfection, DNA sequencing was performed to validate all constructs.Subsequently, using the Lipo3000 transfection reagent for plasmids transfection, recombinant lentiviruses with Flag-tagged DDX17 were obtained from GENECHEM Co., Ltd, Shanghai.pLKO.1 plasmid was used to construct the gene knockdown stable cell lines.A detailed method for the production of lentiviral or retroviral supernatants was described previously. 18riefly, 293T were transfected with the plasmids pMD2.G, psPAX2, shRNA plasmids by Lipofectamine 3000.For the formation of stable cell lines, hepatocytes underwent lentiviral transfection for at least 24 h and selected with 2.5-5 μg/mL puromycin or corresponding resistance medium for at least 7 days.The effect of gene overexpression and knockdown was validated by RT-PCR and WB.Detailed information on plasmids was listed in Table S3.

Histological analysis
The haematoxylin and eosin (H&E) was stained to represent the accumulation of lipid and inflammation status using the liver sections.The NASs were calculated similarly to clinical specimens.Masson trichrome (MT) and Picrosirius red staining (PSR) were performed to visualise the content and type of fibres in the paraffin-embedded sections.The histological features were obtained using the light microscope (Nikon ECLIPSE 80i, Japan).For Oil red O staining, use OTC to embed liver tissue and stain these slides with oil red O working solution, and then these slides were scanned using digital slide scanner (NanoZoomer S360; Hamamastu).For liver tissues immunohistochemistry (IHC), paraffin sections (4 μm thick) were prepared to deparaffinise and rehydrate by xylene and ethanol.Subsequently, endogenous peroxidase was inactivated using the 0.3% hydrogen peroxide.The detailed experimental procedures were executed following the protocol of the ZSGB-BIO kit.The immunohistochemical staining scores were analysed by combining the staining intensity score with the proportion of masculine cells as described previously. 20For the histochemical staining score of the clinical NAFLD patient samples, the tissue sections underwent scoring, considering both the intensity of staining (ranging from 0 to 3 points, corresponding to non-staining, light yellow, light brown and dark brown, respectively) and the extent of positive staining (ranging from 0 to 25, 0 to 25, 26 to 50, 51 to 75 and 76−100%, respectively, rated from 1 to 4 points), and then the two scores were added into the final score.For the histochemical staining score of mouse liver tissue sections, the software Image J was utilised for the quantification of the stained area in the images and the positive staining area was recorded for subsequent analysis.The related primary antibodies are specifically listed in Table S4.

Immunofluorescence analysis
Immunohistochemical fluorescence: Tissue sections were incubated with 10% BSA for 1 h at 37 • C after the antigen retrieval, and subsequently, primary antibodies were applied and left to react overnight at 4 • C. The primary antibodies are listed in Table S4.Then, tissue sections were exposed to goat anti-rabbit immunoglobulin for 1 h.Nuclei were stained by DAPI.Images were acquired using the fluorescence microscope (Pannoramic MIDI; 3DHISTECH) and quantified using Image J software.Immunofluorescence (IF): Cells were fixed with paraformaldehyde after co-transfected with indicated plasmids for 48 h.Then, cells were subjected to incubation with the specified primary antibodies after being permeabilised with 0.5% NP-40 (Nonidet P 40) and blocked with 5% goat serum albumin, and subsequently being incubated fluorescein-conjugated secondary antibodies for 1 h.DAPI is used for staining cell nuclei.Images were obtained under the fluorescent microscope (Nikon Digital ECLIPSE C1, Japan).

Oil Red O staining
Indicated cells were cleaned using 60% isopropyl alcohol, then dyeing with Oil red O working solution for about 10−30 min.Then, the stained cells were rinsed with 75% ethanol and fixed by glycerol after being washed by PBS.

Mouse metabolic studies
During the experiments, body weight (BW) was assessed at different time points, and liver weight (LW) was assessed at the end point.In the glucose tolerance test (GTT) experiment, mice were subjected to a 16-h fast followed by intraperitoneal glucose (1.5 g/kg) injection.And in the insulin tolerance test (ITT) experiment, mice were fasted for 6 h before intraperitoneal insulin (0.5 U/kg) injection.Subsequently, blood glucose levels were measured every half hour continuously for 2 h post-administration for both the GTT and ITT.We utilised the animal monitoring system (Columbus Instruments, USA) to track the energy expenditure of the mice.Over a 48-h period, we recorded metabolic parameters, including oxygen consumption (VO 2 ), carbon dioxide emission (VCO 2 ) and food intake, in a single-chambered enclosure, following established procedures. 21

Mouse serum and liver assay analyses
The levels of serum alanine transaminase (ALT), aspartate aminotransferase (AST), total triglycerides (TG) and total cholesterol (TC) were measured on an Chemray 240 and Chemray 800 (Rayto Life and Analytical Sciences Co., Ltd.) according to the instructions.The hepatic TG and TC contents were extracted and measured with the detection kit (C061 and C063; Changchun Huili Biotech Co., Ltd) according to the guidance.

Western blot analysis
Western blotting analyses were conducted following a standard protocol.The primary antibodies are listed in the Table S4.Three different primary antibodies were used to detect different expression of DDX17.Proteintech (19910-1-AP) was used in the western blot and coimmunoprecipitation (COIP), Abcam (ab70184) was used in human liver in IHC and the Bethyl Laboratories (A300-509A) was used in the livers of mice.Each experiment has been repeated for three times.

RNA isolation and RT-qPCR
Total RNA was isolated from liver tissues and indicated cells using TRIzol™ Reagent and a real-time fluorescent quantitative PCR assay was conducted following its instructions.The exact method and analysis of RT-qPCR was described previously 18 and Table S5 has listed all primer information.

| RNA-sequencing and analysis
The liver tissues from the DDX17-Flox mice and DDX17-CKO mice after MCD administration were lysed in TRI-zol™ Reagent.Total RNA extraction was accomplished using TRIzol™ Reagent from Invitrogen™, followed by a real-time fluorescent quantitative PCR assay to confirm the knockout efficiency.Subsequently, the samples were sent to Novogene Bioinformatics Technology Co. Ltd (Beijing, China) for further processing.The transcriptome sequencing and subsequent analysis were conducted by Novogene.In a concise summary, the PCR products were subjected to purification utilising the AMPure XP system, and the quality assessment of the library was performed through the utilisation of the Agilent Bioanalyzer 2100 system.Genes were considered as differentially expressed if p < .05assessed by DESeq2.Differentially expressed genes (DGEs) were analysed by Gene Ontology (GO) enrichment analysis with the R package.The statistical enrichment of DGEs was acquired by using the cluster Profiler R package in KEGG pathways.Reactome pathways of DGEs (p < .05)were performed by combining the human model species' reactions and biological pathways.GSEA was implemented using GSEA analysis tool, GO, KEGG and Reactome.Related results were listed in Tables S6-S8.

CUT&Tag analysis
The CUT&Tag experiments, including the construction of DNA library, were carried out according to the protocol of hyperactive Universal CUT&Tag Assay Kit (TD903-01/02, vazyme).Briefly, cells were harvested and counted (100,000 cells).The genomic DNA (gDNA) samples were eluted using 26 μL of Millipore water, quantified with the Onedrop system, and then made ready for library amplification.The amplification cycles were 16 according to the index of Illumina (Vazyme#TD202-01) and the products were purified by 1.2× magnetic beads (Vazyme#N411-01), and the final library elution volume was 22 μL.The cDNA libraries underwent sequencing on the Illumina Novaseq 6000, employing 150-bp paired ends and delegated to Novogene Biotech Co., Ltd.(Beijing China) after the size was determined by the Agilent 2100 Tape Station analysis.Detailed analysed results were listed in the final report of biological information analysis (Additional files 3: X101SC21061883-Z01-J001-B1-36)

Absolute quantitative lipidomics
The liver tissues from the DDX17-Flox mice and DDX17-CKO mice after MCD administration were consigned to Applied Protein Technology (Shanghai, China) to perform the lipid extraction and mass spectrometry-based lipid detection.In each group, six replicates were mixed equally as the QC sample.QC samples were conducted to assess the system stability and data reliability during the process.Samples were divided by the UHPLC Nexera LC-30A (SHIMADZU), followed by LC-MS/MS analysis.The lipid identification, peak extraction with alignment and quantification of lipid molecules and internal standard lipid molecules were assessed using the Lipid Search software (Thermo Scientific™).In this experiment, the stability of devices, the repeatability of tests and the reliability of the data were fully evaluated by six quality control items.Detailed data analysis including the lipid class, lipid species, lipid composition, lipid concentration and so on were conducted by Applied Protein Technology and shown in the Additional files 4.

ChIP assays
The ChIP assays were conducted following the protocol outlined in the SimpleChIP R Plus Sonication Chromatin IP Kit (CST, #56383).Before harvested, cells were washed with prechilled PBS, and then followed by incubation in 1% formaldehyde for 15 min at RT for chromatin cross-linking.Then, the cell pellets were lysed in the specific lysis buffer prepared according to the protocol and then sonicated to obtain 200-500 bp DNA fragments.Immunoprecipitations were carried out using anti-DDX17, anti-Histone 3 and normal rabbit IgG.Using RT-PCR to detect the immunoprecipitated DNA fragments with the validated primers, which were predicted and designed for the region of cyp2c29 promoters were listed in Table S5.

Dual-Luciferase reporter assay
Briefly, indicated cells were seeded in a 24-well plate with 5 × 10 4 per well and cultured for 24 h.Subsequently, we performed a co-transfection of the promoter reporter plasmids (0.48 μg) and the pRL-TK plasmids (0.02 μg) into the cells using Lipofectamine 3000 from Invitrogen.After 6 h, the culture medium was changed with fresh medium for 48 h.Then, we quantified luciferase activity utilising a GloMax 20/20 Luminometer manufactured by Promega.
To ensure accuracy and consistency, we normalised the luciferase activity to the Renilla activity by following the prescribed protocol of the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
The following steps were conducted following a standard protocol.

Statistical analysis
We conducted statistical analysis by employing GraphPad Prism software.To compare two sets of data, we utilised a two-tailed Student's t-test.For the comparisons involving multiple groups, we executed a one-way ANOVA.Quantitative data were analysed using Spearman's correlation when applicable.Statistical significance was established as follows: *p < .05;**p < .01;***p < .001.

DDX17 expression is enhanced as NASH progresses
Hepatic DDX17 protein levels were elevated in patients with NAFLD or NASH than in those with non-steatosis.Furthermore, compared with the non-steatosis group, the NAFLD and NASH group demonstrated substantially higher DDX17 expression (Figure 1A) in the quantification of IHC.To prepare for DDX17 causation studies, we explored DDX17 expression in various NASH mouse models.Consistent with results in human subjects, the levels of DDX17 protein and mRNA were significantly up-regulated in the livers of HFD-fed, MCD-fed and ob/ob mice during NASH progression (Figures 1B-F and S1A-D).Then, L02 cells were stimulated by PAOA to investigate the mechanism underlying the up-regulation of DDX17 expression.We found that PAOA (PA, 0.25 mM; OA, 0.5 mM) treatment increased the mRNA and protein expression levels of DDX17 in L02 cells (Figures 1G-I and S1E).The findings from this study indicate a potential association between DDX17 and NASH and DDX17 potentially exerting a crucial influence on its in its progression.

DDX17 promotes hepatic steatosis in murine NASH models
Next, we infected L02 cells with lentivirus DDX17 overexpression and DDX17-knockdown clones (Figures S2A  and 2E) in order to investigate how DDX17 impacts lipid metabolism in hepatocytes.Oil Red O staining, cellular TG and cellular TC analysis showed a distinct increase in lipid accumulation induced by PAOA in the DDX17overexpressing group and a significant decrease in the DDX17 knockdown group compared with the control group.There were no statistically significant distinction observed between the two groups when exposed to BSA (Figures S2B-D and S2F-H).In order to assess the distribution of DDX17 in the liver, we first performed bioinformatic analyses of single-cell RNA sequencing (scRNA-seq) data of the human liver (GSE115469) to detect the characteristics of DDX17 expression in human liver cells.The results indicated that in a normal liver, the expression of DDX17 is low to moderate in hepatocytes, Kupffer cells, hepatic stellate cells (HSCs), endothelial cells, cholangiocytes, erythroid cells, T-cells and B-cells (Figure S3A).In order to investigate the function of hepatocyte DDX17 in NASH in vivo, we constructed hepatocyte-specific DDX17-knockout mice (DDX17-CKO) and subjected them to a chow diet or a HFD for 24 weeks.Subsequently, the mice underwent metabolic assessments utilising a Comprehensive Lab Animal Monitoring System.Comparing the DDX17-CKO mice with the DDX17-Flox mice, differences observed in terms of oxygen consumption (Figure S3B), carbon dioxide emission (Figure S3C) or food intake (Figure S3D) were not significant.
After 24 weeks of HFD consumption, the CKO-HFD group exhibited a significant reduction in BWs, LWs and liver-to-BW ratios as compared with the Flox-HFD control group (Figures S4A, 2A and B).The GTT demonstrated improved glucose tolerance, while the ITT indicated less insulin resistance in CKO-HFD mice compared with Flox-HFD mice (Figures 2C and D).In CKO-HFD mice, the serum concentrations of ALT, AST, TG and TC were found to be lower in comparison with those observed in Flox-HFD mice (Figures 2E-H).Furthermore, Flox-HFD mice shown more severe steatosis in the liver than CKO-HFD mice, which was supported by hepatic TG and TC concentrations (Figures 2I and J), H&E and Oil Red O staining (Figures 2K and S4B).These findings imply that DDX17 may contribute to the accumulation of lipids in hepatocytes and promote NASH progression in mice.

Hepatocyte-specific DDX17 deficiency alleviates MCD-induced NASH
To further validate the role and precise mechanism of hepatic DDX17 in the development of NASH, we subjected DDX17-Flox and DDX17-CKO mice to a MCD diet for 16 weeks, which were respectively labelled as Flox-MCD and CKO-MCD mice for short.After these 16 weeks, the CKO-MCD mice showed significantly lower BWs, LWs and liver-to-BW ratios than the Flox-MCD controls (Figures S5A, 3A and B).Significantly higher oxygen consumption (Figure S5B), carbon dioxide emission (Figure S5C) and food intake (Figure S5D) were noted in the DDX17-CKO mice compared with those of DDX17-Flox mice.The levels of ALT, AST, TG and TC in the liver were decreased in CKO-MCD mice compared with Flox-MCD mice (Figures 3C-F).Furthermore, Flox-MCD mice exhibited more severe steatosis in the liver than CKO-MCD mice, as supported by the concentrations of hepatic TG and TC (Figures 3G and H) as well as H&E and Oil Red O staining (Figures 3I-L).

AAV8-mediated hepatic DDX17 overexpression aggravates MCD-induced NASH
To further validate the role of hepatocyte DDX17 in the development of NASH, we overexpressed DDX17 in 6week-old mice by injection of an adeno-associated virus expressing Ddx17 (AAV8-Ddx17) in parallel with its control followed by MCD feeding for another 16 weeks (Figure 4A).After these 16 weeks, the AAV8-Ddx17 mice exhibited notable increased LWs and liver-to-BW ratios in comparison with the AAV8-Con mice (Figures 4B and C).The AAV8-Ddx17 mice exhibited elevated serum levels of ALT, AST, TG and TC in comparison with the AAV8-Con mice within the liver samples (Figures 4D-G).Furthermore, AAV8-Ddx17 mice displayed a more pronounced liver steatosis than AAV8-Con mice, as supported by the concentrations of hepatic TG and TC (Figures 4H and I) as well as H&E and Oil Red O staining (Figures 4J-M).These findings suggest that DDX17 could be involved in lipid accumulation in hepatocytes and promote the NASH progression in an MCD-induced mouse model.that had been fed a MCD was conducted and the DEGs are displayed in volcano plots (Figure 5A).GO function enrichment analysis revealed that epoxygenase activity (arachidonic acid [AA] monooxygenase activity, oxidoreductase activity, etc.), related to metabolic pathways (xenobiotic stimulus, epoxygenase P450 pathway), inflammation (immune response, cytokine production) and extracellular matrix organisation, may be associated with the progression of NASH (Figures 5B and S6A).To further analyse the RNA-seq results, we identified several lipid metabolism pathways, such as AA metabolism, retinol metabolism, steroid hormone biosynthesis and linoleic acid metabolism using GSEA analysis and a heatmap of the DEGs (Figures 5C and D).Ultimately, 10 CYP450 family genes with significant difference were obtained through Venn diagram analysis (Figure 5E) and validated by RT-PCR (Figure 5F).Furthermore, the epoxygenase P450 pathway and positive regulation of cytokine production were further demonstrated via directed acyclic graph analysis of the differentially up-regulated and down-regulated genes in the two groups (Figures S6B-4D).

DDX17 regulates lipid metabolism and the subsequent inflammatory response by regulating the CYP450 family genes
Based on the RNA-seq analysis, our findings suggest that DDX17 may play a role in lipid metabolism and in regulating the progression of inflammation by regulating the CYP450 family genes in MCD-induced NASH.

DDX17 transcriptionally repressed Cyp2c29 gene expression by cooperating with CTCF and DDX5 in L02 hepatocytes
Given the potential role of DDX17 in inhibiting the expression of CYP450 family genes, we conducted a CUT&Tag of DDX17 in L02 cells treated with PAOA.The genomewide peak annotation of DDX17 indicated that the highest proportion of the promoter within 1 kb (Figures 6A, B  and S7A) and the metabolic pathways and NAFLD were enriched in the pathway enrichment analysis of differential peak between IgG and DDX17 groups (Figure S7B).Next, the bound motifs of DDX17, which were identified through HOMER known motifs, were shown (Figure 6C) and found to contain 'CCCTC', which has been previously reported to be the classic CTCF motif and is shared with the DDX17 paralog, p68 (DDX5). 22,23Thus, we hypothesised that DDX17 may bind to the CTCF motif in order to transcriptionally regulate Cyp2c29 gene expression, which was found to be the most significantly up-regulated gene in the DDX17-CKO group.We then performed the ChIP assay to prove that DDX17 binds to the promoter of Cyp2c29, especially the first 'CCCTC' motif close to exon 1 (Figures 6D-E  and S7C), and the relative luciferase activity of the wildtype or sites-mutated Cyp2c29 promoter was respectively detected in indicted L02 cell groups (Figure 6F).
Previous studies have shown that CTCF regulates key aspects of gene expression, including transcription activation and repression. 23,26Thus, we hypothesised that DDX17 and its paralog, DDX5, may cooperate with CTCF to regulate the transcriptional repression of Cyp2c29.Therefore, we conducted further ChIP assays and identified a decreasing interaction between DDX17 and the Cyp2c29 promoter after knock-down of CTCF or DDX5 via siRNA transfection in L02 cells (Figures 7A and B).According to the above data, we speculated that DDX17 interacts with CTCF and DDX5 to function as a co-TF.Furthermore, the colocalisation and interaction of DDX17 with CTCF or DDX5 were identified using IF and COIP assays in 293T cells (Figures S8A-C).We then conducted a series of luciferase reporter assays in L02 cells and 293T cell with the indicated treatment.We found that CTCF or DDX5 could cooperate with DDX17 to repress the Cyp2c29 luciferase reporter (Figures 7C, D and S8D-G).In addition, the corresponding mRNA and protein levels of CYP2C19 and CYP2C9, 27,28 which are reported as the human orthologs of Cyp2c29 in mice, were altered, which is consistent with the results of the luciferase reporter assays (Figures 7E-L).
Our analysis of RNA-seq and CUT&Tag, combined with ChIP and luciferase reporter assays, indicated that DDX17 might cooperate with CTCF and DDX5 in order to regulate the transcriptional repression of Cyp2c29 gene expression in mice and hepatocytes.

DDX17 promotes NASH by repressing cyp2c29 gene expression in mice and hepatocytes
Based on the abovementioned results, our next objective was to authenticate the mechanism through which DDX17 governs the expression of the cyp2c29 gene in both human and mouse liver tissues.We first verified that the protein and mRNA expression of cyp2c29 were considerably downregulate with the progression of NASH in the liver of HFDor MCD-induced mice and ob/ob mice (Figures S9A-E).This observation aligns with prior research findings. 29,30e conducted additional investigations into its expression in the livers of individuals who do not have steatosis, as well as in patients with NAFLD and NASH.We found that hepatic CYP2C19 (the human ortholog of Cyp2c29) protein levels were notably reduced in the liver of individuals with NAFLD or NASH compared with those with nonsteatosis livers (Figure 8A).We also observed a notable negative correlation between DDX17 and CYP2C19 in the livers of patients (Figure 8B).Furthermore, we identified a higher Cyp2c29 protein and mRNA expression in the liver of DDX17-CKO mice than in Flox mice after MCD administration (Figures 8C and 5F).In line with the outcomes F I G U R E 5 DDX17 may alter the landscape of lipid metabolism, inflammation and fibrosis by regulating the expression of CYP450 family genes in murine NASH.(A) Volcano plots indicating the DEGs (red, down-regulated genes; yellow, up-regulated genes) between DDX17-Flox and DDX17-CKO mice were fed with MCD for 16 weeks.(B) GO function enrichment analysis of differential up-regulated genes in MCD-induced steatohepatitis in hepatocyte-specific DDX17 knockout mice.Down/up-regulated genes enriched in pathways were indicated (black font, down; red font, up).(C) GSEA analysis of up-regulated genes in DDX17-CKO mice were mainly enriched in four metabolic pathways (arachidonic acid metabolism, linoleic acid metabolism, retinol metabolism and steroid hormone biosynthesis).(D) Heatmap analysis of up-regulated and down-regulated genes between DDX17-Flox and DDX17-CKO group.(E) Venn diagram analysis of genes in four differential metabolism related pathways was conducted to obtain ten CYP450 family genes.(F) Relative mRNA levels of ten CYP450 family genes in the livers of MCD-fed DDX17-CKO and DDX17-Flox (**p < .01,***p < .001;n.s., not significant).For (F), statistical analysis was performed using the two-tailed Student's t-test.All data are shown as the mean ± SD.   observed in vivo, CYP2C19/C9/C8 (the human orthologs of Cyp2c29) mRNA and protein levels were increased in the L02-shDDX17 group but markedly decreased in the L02-DDX17 group after PAOA treatment (Figures 8D-G).In addition, we detected a higher expression of Cyp2c29 in the liver of DDX17-CKO mice than in Flox mice after MCD administration (Figure 8H).Subsequently, we also found that Cyp2c29 was down-regulated and demonstrated a significant negative relationship with DDX17 in the livers of HFD-or MCD-diet-fed mice over time (Figures S9F-I).Meanwhile, we validated that Cyp2c29 deficiency promotes the lipid accumulation in L02 cells (Figures S10A-D).These results suggest that DDX17 may promote NASH progression by transcriptionally repressing Cyp2c29 gene expression.

DDX17 alters lipid composition in murine NASH
As we know, metabolic dysfunction such as hepatic steatosis is regarded as a significant initial stage in the development of NASH.Lipid accumulation and composition not only constitute a first hit in the progress, but inappropriate lipid metabolism might also drive key further steps such as inflammation and fibrosis in NASH. 31yp2c29 has been demonstrated to contribute to fatty acid metabolism and inflammatory response in fatty liver. 32,33n order to further investigate the effects of DDX17 in lipid metabolism in NASH, we performed an absolute quantitative lipidomics analysis in liver tissues from DDX17-CKO mice and DDX17-Flox mice after MCD administration.A total of 20 lipid classes and 1811 lipid species were quantified, including various glycerolipid classes such as monoradylglycerols (MGs), diradylglycerols (DGs) and triradylglycerols (TGs), along with sphingolipid classes like ceramide and sphingomyelin (Figure S11A).The lipid subgroup composition and dynamic distribution range of lipid content were analysed in DDX17-CKO and DDX17-Flox mice after MCD administration, as shown in Figures 9A  and S11B.Furthermore, lipid content was detected and analysed at a subgroup-and molecular level, along with the total lipid content.We found that the total lipids and the main compartment involved glycerolipids (primarily TGs) with considerable amounts of MGs and DGs, which were markedly different between the two groups (Figures 9B-E  and S11C-F).Based on the univariate analysis, all detected lipid molecules were analysed.The outcomes were graphically represented through a volcano plot, as illustrated in Figure 9F.Additionally, we applied each sample to hierarchical clustering analysis using the lipids with significant differences (VIP > 1, p value < .05)(Figure 9G).These lipid molecules are listed in Additional files 3. Lipid chain and saturation analyses of TGs, DGs and MGs were also conducted between the two groups (Figures 9H-I and S11G-J).It is well known that the lipid chain length and saturation of lipid play a role in regulating the biological functions of the cell, such as unfolded protein response 34,35 and other membrane signals. 36,37Furthermore, lipid composition may influence the extracellular environment or cells such as macrophages. 38Collectively, our lipidomic analysis revealed the effect of DDX17 on liver lipid metabolism in response to MCD feeding, which may support the function of DDX17 in fatty acid metabolism mediated by Cyp2c29.

DDX17 promotes the progression of liver inflammation and liver fibrosis in murine NASH model
To detect inflammation activation and fibrosis in our murine NASH model, inflammatory and fibrosis-related genes were further validated using RT-PCR (Figures 10A  and S12A-C).Fibrotic markers, such as Collagen I, transforming growth factor beta (tgf-β) and α-smooth muscle actin (α-SMA), were detected in liver tissues from DDX17-Flox and DDX17-CKO mice groups (Figure 10B).Furthermore, we found that a-SMA and Collagen, as well as MT, PSR and Sirius red-stained areas were significantly downregulated in the DDX17-CKO mice group in comparison with the DDX17-Flox mice group (Figures 10C and S12D).
The above RNA-seq analysis suggests that immune response, cytokine production and extracellular matrix organisation may participate in inflammation progress and fibrosis, respectively.Therefore, using the IHC/multiplex IF (IHC/mIF), we assessed the expression of F4/80 (a marker for macrophages), CD86 (a marker for M1 macrophages) and CD206 (a marker for M2 macrophages) in the two experimental groups (Figures 10D and E).Differential quantification revealed that F4/80 and CD86 were dramatically decreased in DDX17-CKO mice compared with DDX17-Flox mice.Recent, studies 39 on macrophages in NASH progression have identified NASH-associated macrophages (NAMs), which notably possess a distinct array of molecular markers, including Gpnmb, Trem2, C1qa and Apoe, which were identified via our analysis of bulk RNA-seq and validated by RT-PCR (Figure 10F).Furthermore, our discovery revealed a substantial downregulation in the protein levels of p-STAT1 and p-STAT6 levels were significantly up-regulated in the liver tissues of MCD-fed DDX17-CKO mice when compared with DDX17-Flox mice (Figure 10G).Relative mRNA levels (Figure S12E) of pro-inflammatory cytokines (Tnfa, Il6, Il1b and Cxcl10) and serum ELISA levels of pro-inflammatory cytokines IL-6 and TNF-a (Figures S12F and S12G) were also significantly inhibited in the livers of MCD-fed DDX17- CKO mice when compared with DDX17-Flox mice. 40What is more, we validated that the levels of DDX17 in AML12 shown no effect on RAW264.7 (Figures S13A and S13B).By integrating RNA-seq and lipidome analyses, our hypothesis suggests that DDX17 could potentially be involved in governing lipid metabolism and M1 macrophage activation in our murine NASH model.
Overall, we identified that DDX17 depletion alleviated MCD-induced NASH, and it might play a role mechanistically in the progression of inflammation and fibrosis through the regulation of lipid metabolism and the activation of M1 macrophages.

3.10
Cyp2c29 mediates the function of DDX17 in lipid accumulation and macrophage activation Next, we explored whether DDX17 promotes lipid accumulation and inflammatory responses by regulating the expression of Cyp2c29.We found that overexpression of Cyp2c29 diminished lipid accumulation induced by PAOA in both the L02-shDDX17 and L02-DDX17 groups when compared with their respective controls (Figures 11A and  B).Furthermore, we also found that the mRNA levels of activated M1 macrophage markers including CD86, TNF-α, iNOS and IL-6 were markedly decreased in the AML12-DDX17 group after overexpression of Cyp2c29 (Figure 11C).
Following the findings outlined above, we proceeded to investigate the correlation between the activation of M1 macrophages and DDX17 and CYP2C19 in the livers of human subjects.The quantification of mIF revealed an elevation in iNOS, a marker associated with M1 macrophages, and a reduction in CD163, a marker for M2 macrophages, within the livers of individuals afflicted with NAFLD or NASH, as opposed to those with non-steatotic livers (Figure 12A).In addition, DDX17 was significantly positively correlated with iNOS (r = 0.6257, p < .0001)and negatively correlated with CD163 (r = −0.8007,p < .0001),and CYP2C19 was significantly negatively correlated with iNOS (r = −0.6034,p < .0001)and positively correlated with CD163 (r = 0.5908, p < .0001)(Figures 12B-E).These results further proved that DDX17 plays a role in lipid metabolism, activation of M1 macrophages and subsequent inflammatory responses through the regulation of Cyp2c29 during the progression of NASH.

DISCUSSION
In this research, we identified that DDX17 in hepatocytes is a novel NASH promoter that stimulates lipid accumulation, inflammation and fibrosis in the liver by cooperating with DDX5 and CTCF in order to repress the transcription of Cyp2c29, which ultimately diminishes the production of 14,15-EETs.In vivo studies have shown that hepatocyte-specific knockout of DDX17 attenuates MCDor HFD-diet-induced hepatic steatosis, inflammation and fibrosis.Mechanistically, we found that hepatic DDX17 affects lipid accumulation by repressing the transcription of Cyp2c29, which metabolises AA to several beneficial metabolites such as EETs and specialised proresolving mediators (SPMs). 32,33,41,42CUT&Tag analysis, ChIP and luciferase reporter assays were conducted to explore the transcription of DDX17, and a series of rescue experiments were performed by supplementing with 14, 15-EET and its antagonist to confirm the role of DDX17/Cyp2c29/ 14, 15-EET pathway in lipid metabolism and inflammation.Hence, our investigation unveils a fresh mechanism, shedding light on the DDX17 targets within the context of NASH, thus offering a promising avenue for NASH treatment.DDX17 (p72), and its paralog DDX5 (p68), which are members of the RNA helicase family, can interact with several components of transcriptional machinery such as RNA polymerase II, histone deacetylases and CBP/p300 5,13,14 in order to play the roles in transcription.
6][17] In our previous work, 18 we found that DDX17 was up-regulated in the livers or HCC tissues of DEN treated C57 mice that had been fed a HFD diet for 8 months compared with mice simply treated with DEN alone.Consequently, our conjecture postulates the role of DDX17 in the progression of both NASH and NASH-HCC.
In our present study, hepatocyte-specific knockout of DDX17 attenuated MCD or HFD-diet-induced hepatic steatosis, inflammation and fibrosis.Our analysis of RNAseq and CUT&Tag, combined with ChIP and luciferase reporter assays, indicated that DDX17 transcriptionally represses Cyp2c29 gene expression by cooperating with CTCF and DDX5.Using absolute quantitative lipidomics analysis, we found that a hepatocyte-specific DDX17 deficiency decreased lipid accumulation and altered lipid composition in the livers of mice after MCD administration.Combined with RNA-seq analysis and lipidome analysis, we found that DDX17 may play a role in the regulation of lipid metabolism and M1 macrophage activation in murine NASH models.Furthermore, a global RNA landscape was detected in the livers of MCD-administered DDX17-CKO mice and DDX17-Flox mice using RNA-seq.Bulk RNA-seq revealed the role of DDX17 in lipid metabolism, inflammation and fibrosis.Furthermore, differential lipid metabolism and macrophage activation-related genes were analysed and visualised to identify the specific mechanisms.Among them, NAMs signature markers, such as Gpnmb, Trem2, C1qa and Apoe, 39 were also identified via our bulk RNA-seq and related analysis.This result implies that DDX17 could potentially influence hepatic macrophage activation and may even promote the progression of NASH-HCC by remodelling the tumour-prone liver microenvironment.Moreover, we demonstrated that DDX17 may promote the activation of M1 macrophages and inhibit the activation of M2 macrophages by causing the metabolic disorders.Recently, Deng et al. 43 reported that M2 macrophages possess the capacity to suppress stellate cells activation by secreting exosomes containing microRNA-411-5p.We found that differential activation of macrophages may promote the advancement of liver inflammation and fibrosis in murine NASH models.These results indicate that pro-inflammatory genes and fibrotic genes were significantly down-regulated in the liver tissues of DDX17-CKO mice.Thus, DDX17 may not only regulate lipid metabolism in hepatocyte, but also indirectly regulate the activation of M1 macrophages and the inhibition of M2 macrophages, as well as subsequent HSCs activation.We combined RNA-seq analysis in murine NASH liver and CUT&Tag in L02 cells with ChIP and luciferase reporter assays in order to validate the transcriptional regulation of Cyp2c29 by DDX17.Consistent with previous studies, [22][23][24] our results indicated that DDX17 might cooperate with DDX5 and CTCF to regulate the transcription of Cyp2c29 by binding the classic CTCF motif "CCCTC" at the promoter of Cyp2c29.In our experiments, we cannot exclude the possibility that other TF or RNA polymerase can interact with DDX17, DDX5 and CTCF to regulate the transcription of Cyp2c29.Therefore, more specific binding modes and epigenetic regulations should be elucidated.
Cyp2c29, the mouse ortholog of both CYP2C9 and CYP2C19, belongs to the CYP2C subfamily and cytochromes P450 superfamily (CYP450s). 29,30The CYP2C subfamily in humans comprises four isoforms (CYP2C19, CYP2C9, CYP2C8 and CYP2C18) that account for approximately 20% of the total liver P450 contents. 44hey play a critical role in the oxidative metabolism of xenobiotics and endogenous substrates.Recent research findings have indicated that Cyp2c29 plays a critical role in the metabolism of AA, leading to the formation of bioactive EETs.These compounds have demonstrated significant anti-inflammatory properties and exhibit protective effects in a range of conditions, encompassing NASH and cardiovascular diseases. 29,30,45,46In our current investigation, the mRNA and protein expression of Cyp2c29 tended to decrease with NAFLD progression, which is consistent with our results in the HFD-and MCD-induced NASH models (Figures S7A-H).Collectively, these studies indicate that the inhibition of EET biosynthesis mediated by the hepatic CYP450s, a pivotal inflammatory risk factor in the fatty liver disease, and that the CYP epoxygenase pathway is a primary regulator of the inflammatory response in the progression of NAFLD/NASH.8][49][50] Based on the above studies, we performed a series of rescue experiments in which overexpression of Cyp2c29 or supplementation with 14,15-EET alleviated lipid accumulation and macrophage activation in L02 and AML12 cells.Furthermore, we found negative relationship between DDX17 and Cyp2c29 expression in HFD-and MCD-induced murine NASH models and in patients with NASH.Overall, these findings suggest that the down-regulation of Cyp2c29 expression by DDX17 and the subsequent low EET levels may trigger lipid metabolism dysfunction and anti-inflammatory effects and may be a critical pathological consequence of NASH in vivo.
NAFLD is a complex disease with highly heterogeneous factors and clinical manifestations among individuals.The 'multiple-hits' hypothesis involved various risk factors provides a more acceptable interpretation of the pathogenesis of NAFLD.In addition to genetic factors, inflammation, innate immunity, metabolic homeostasis, lipotoxicity and cell death have been found to be related to NASH. 1,3ecently, it is reported that DDX17 has a protective role in hepatocytes when confronted with lipid accumulation induced by oleic acid and palmitic acid. 51In view of the differences, we considered the following factors.First, our study chosen the normal liver cell line (L02), and a human and mouse hepatic carcinoma cell line (HepG2 and Hep1-6) were used in the literature, which may be due to the differences in the genetic background and metabolic mode between normal liver cells and tumour cells.In addition, the mechanism of DDX helicase family is very complex in different cell and disease background.DDX helicase regulate the expression of target gene by binding to different TF or chromatin complexes.In view of such a precise regulatory mechanism, it is difficult to accurately simulate the physiological function of DDX helicase by simply performing the experiments in vitro.Based on these factors, we adopt the hepatocyte-specific DDX17 knockout mice and two high-fat mice models to explore the metabolic phenotype and molecular mechanism, which may make up for the shortcomings of cell-line models.In this study, we demonstrated that DDX17 promotes lipid accumulation in hepatocytes.The mRNA and protein levels were markedly up-regulated in the progression of HFDand MCD-induced NASH in mice.However, there are still some shortcomings, such as the therapeutic effects of DDX17 in NASH mice and the exclusion of other metabolites of Cyp2c29, which remain to be solved.What is more, the MCD diet model, which was chosen in our experiments and further analyses, exists several limitations during to causing the acute weight loss and without the insulin resistance.Therefore, reasonable NASH models should be conducted to validate our results in the future.
In conclusion, our findings revealed a substantial increase in DDX17 expression within the livers of both human subjects and murine models with NASH.Further, hepatic DDX17 transcriptionally represses Cyp2c29 gene expression by cooperating with CTCF and DDX5.
The results indicate that DDX17 contributes to the progression of NASH by stimulating the lipid accumulation in hepatocytes and inducing the activation of M1 macrophages as well as promoting a subsequent inflammatory response and fibrosis through the transcriptional repression of Cyp2c29 in mice.Overall, our study reveals the role of the DDX17/Cyp2c29/ 14, 15-EET pathway in lipid metabolism and subsequent inflammatory response and provides promising targets for the treatment of hepatic steatosis, inflammation and fibrosis.

A C K N O W L E D G E M E N T S
We are grateful to those patients for donating their liver tissues for the studies.We would like to thank Editage (www.editage.cn)for English language editing.This work was supported by The National Natural Science Foundation of China (No. 8187111473, 8217113337 to Wanguang Zhang) and State Key Project on Infection Disease of China (No. 2018ZX10723204-003 to Bixiang Zhang).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data presented in this study are available on reasonable request from the corresponding author.

E T H I C S S TAT E M E N T A N D C O N S E N T T O PA R T I C I PAT E
All animal experiments were conducted in accordance with the protocols were approved by the Tongji Hospital Animal Care and Use Committee (TJH-202101106).All procedures involving human samples were approved by the Tongji Hospital of Huazhong University of Science and Technology Review Board, Wuhan, China and were consistent with the principles outlined in the Declaration of Helsinki.Informed consent was written by the subjects or immediate families of the liver donors.

F I G U R E 3
Hepatocyte-specific DDX17 deficiency alleviates MCD diet-induced hepatic steatosis.(A and B) Liver (A) and liver weight/body weights (B) of Flox-MCD and CKO-MCD mice were fed with MCD for 16 weeks.(C-F) Serum ALT (C), serum AST (D), serum TG (E) and TC (F) levels from Flox-MCD and CKO-MCD mice that were fed with MCD for 16 weeks.(G and H) Hepatic TG (G) and hepatic TC (H) levels from Flox-MCD and CKO-MCD mice fed with MCD for 16 weeks.(I-L) Representative images (left) and quantitative results (right) of H&E-stained (I) and Oil Red O-stained (K) liver sections from Flox-MCD and CKO-MCD mice fed with MCD for 16 weeks (red bar, 100 μm; black bar, 100 μm).For the above analysis, Flox-MCD group (n = 7 mice/group) and CKO-MCD group (n = 5 mice/group) were compared (*p < .05;**p < .01;***p < .001).In all statistical plots, data are statistically analysed using the two-tailed Student's t-test and shown as the mean ± SD.

F I G U R E 6
DDX17 transcriptionally repress the Cyp2c29 gene expression by binding the classic CTCF motif of its promoter.(A) A schematic diagram of CUT&Tag analysis in L02 hepatocytes treated with PAOA.(B) Genome-wide peak annotation of DDX17 showing the proportion of exon, intron, promoter, TSS and intergenic region were shown in the pie chart.(C) DDX17 bound motifs identified by Homer known motifs in L02 cells.The motif sequence is shown in the first column, the corresponding transcription factor (TF) is shown in the second column, the p value is shown in third column and the proportion of target sequences and background sequences with motif are shown in the last two column.(D) The predicted binding sites of DDX17 and CTCF.(E) The ChIP of L02 cells show the binding sites (***p < .001;n = 3 independent experiments).(F) The relative luciferase activity of wild-type (left panel) or the sites-mutated (right panel) Cyp2c29 promoter was respectively detected in indicted cell groups (**p < .01;n.s., not significant; n = 3 independent experiments).For (E) and (F), statistical analysis was performed using the two-tailed Student's t-test.

F I G U R E 7
DDX17 down-regulate the gene expression of Cyp2c29 through cooperating with CTCF and DDX5 in L02 hepatocytes.(A and B) The ChIP of L02 cells transfected with CTCF (A) or DDX5 (B) siRNA (n = 3 independent experiments).(C and D) The relative luciferase activity of Cyp2c29 promoter was detected in indicted cell groups in L02 cells respectively transfected with CTCF (C) or DDX5 (D) plasmid.(E-H) Relative mRNA levels of CYP2C19, CYP2C9 and DDX17 in indicated cell group in L02 hepatocytes.(I and J) Representative western blots (left) and quantification (right) of CYP2C19, Flag-CTCF and DDX17 in indicated cell group in L02 hepatocytes.(K and L) Representative western blots (left) and quantification (right) of CYP2C19, Flag-DDX5 and DDX17 in indicated cell group in L02 hepatocytes.*p < .05;**p < .01;#p < .001;n.s., not significant, that was compared with the control group.Each experiment was repeated three times.For (A) and (B), statistical analysis was performed by two-tailed Student's t-test.For (C)-(L), statistical analysis was performed by one-way ANOVA.

TA B L E 1
Correlations with DDX17 in patients.